Collection and therapeutic use of stem cells is among the most rapidly-developing fields of modern medicine. Ability of stem cells to differentiate into blood lineages is the basis of therapies in many hematological disorders and other medical applications. As of now, only hematopoietic stem cells have been proven to provide therapeutic effect. One of the main problems is the difficulty in obtaining sufficient amounts of stem cells. Therefore, supply of stem cells, including cord blood stem cells is limited, and novel sources are in high demand.
Two major directions in the field of augmentation of stem cells collections could be outlined: novel approaches to stem cell mobilization from known sources, and use of novel sources of stem cells. Most optimal is combination of these two approaches which is the main subject matter of this invention.
Hematopoietic stem cells (HSCs), are multipotent stem cells that give rise to all the blood cell types from the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The hematopoietic organs contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. HSCs are a heterogeneous population with different properties, capacities and markers. Many of these markers belong to the cluster of differentiation series, like: CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit;—the receptor for stem cell factor. The HSCs are negative for the markers that are used for detection of lineage commitment.
Mobilization of stem cells is an important step in stem-cell based therapies, especially in treatment of hematological disorders. In adult donors mobilization of hematopoietic stem cells requires puncture of the iliac crest of healthy people who are donating bone marrow. Currently, in clinical practice most often mobilization is performed by administering to the donor growth factors like G-CSF, which activates a cascade of enzymes and complement and releases HSC from its niche. This allows for hematopoietic stem cells to be collected from the peripheral blood.
In general, numerous pharmacological agents mobilize stem cells, ranging from cytokines and growth factors, hormones, to beta-glucans. They differ in their mechanisms of action, some of which are not precisely known, and exert a plethora of effects on both HSCs and their niches. Therefore, pharmacological approach to HSCs mobilization is in general non-specific, and could involve multiple pharmacological agents or their combinations. Stem cells which participate in hematopoiesis are believed to reside in niches in bone marrow in adults. They are hold in place by interaction of multiple adhesion molecules present on the cell surface and surrounding extracellular matrix, or adjacent cells. For example, integrins are molecules that intervene attachments between a cell and the tissues/cells. They also play a role in cell signaling and regulate cellular motility.
There are many types of integrins, and HSCs have multiple types of integrins on their surface. In addition to integrins, other surface proteins such as cadherins, immunoglobulin superfamily cell adhesion molecules, selectins and syndecans mediate cell-cell and cell-matrix interaction and communication. Integrins are heterodimer transmembrane receptors for the extracellular matrix. Natural integrin ligands include laminin, fibronectin, and vitronectin, but they also include fibrinogen and fibrin, thrombospondin, MMP-2, and fibroblast growth factor. Integrin subunits cross the plasma membrane and have short cytoplasmic domains. The molecular mass of the integrin subunits can vary from 90 kDa to 160 kDa. The attachment of the cell is due to formation of multiple cell adhesion complexes, which consist of integrins and many cytoplasmic proteins such as talin, vinculin, paxillin, and alpha-actinin. These proteins regulate kinases such as FAK (focal adhesion kinase) and Src kinase. Adhesion complexes attach to the actin cytoskeleton. Integrins bind ligands by recognizing amino acid stretches on exposed loops, particularly the RGD sequence. Following ligation, integrins mediate signaling events, alone or in combination with growth factor receptors, regulating cell adhesion, and migration by activating canonical pathways, such as integrin-linked kinase (ILK), protein kinase B (PKB/Akt), mitogen-activated protein kinase (MAPK), Rac or nuclear factor kappa B (NF-B). Several classes of integrin inhibitors are currently investigated or used: monoclonal antibodies targeting the extracellular domain of the heterodimer (Vitaxin; MedImmune, Gaithersburg, Md.), synthetic peptides containing an RGD sequence (cilengitide; Merck KGaA, Darmstadt, Germany), and peptidomimetics (S247; Pfizer, St Louis, Mo.), which are orally bioavailable nonpeptidic molecules mimicking the RGD sequence. Natalizumab (Tysabri; Biogen/Idec, Cambridge, Mass., USA) is a recombinant humanized neutralizing IgG4 monoclonal antibody that binds to the (α4-subunit of the α4β1 (VLA-4) and α4β7 integrins. Natalizumab is approved by the FDA for the treatment of Crohn's disease and relapsing Multiple Sclerosis and is postulated to function in these conditions by inhibiting the transmigration of leukocytes through the blood-brain barrier. Following anti-α4 integrin antibody administration, natalizumab-treated MS patients display a rapid and sustained increase in circulating HSPCs (M P Rettig, G Ansstas and J F DiPersio Leukemia advance online publication2 September 2011; doi: 10.1038/leu.2011.197 Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4). Small-molecule antagonists of α4 integrins is an another way to mobilizing HSPCs. BIO5192 is (2(S)-{[1-3,5-dichloro-benzenesulfonyl)-pyrrolidine-2(S)-carbonyl]-amino}-4-[4-methyl-2(S)-(methyl-{2-[4-(3-o-tolykureido)-phenyl]acetyl}-amino)-pentanoylamino]-butyric acid), a potent (Kd of <10 pM) and highly selective small-molecule inhibitor of both the unactivated and activated forms of α4β1 integrin (P Ramirez, M P Rettig, G L Uy, E Deych, M S Holt, J K Ritchey and J DiPersio. BIO5192, a small molecule inhibitor of VLA-4, mobilizes hematopoietic stem and progenitor cells. Blood, Prepublished online Jul. 1, 2009 doi:10.1182/blood-2008-10-184721).
Stahle and Goodman (WO 2001/010841 Fluorene derivatives as integrin inhibitors) describe fluorine derivatives which can be used as integrin inhibitors for the prophylaxis and treatment of blood disorders and disorders propagated through angiogenesis. U.S. Pat. No. 5,912,266 (Beta integrin cell adhesion molecule inhibitors) describes chemical compound as integrin inhibitor. U.S. Pat. No. 6,849,639 (Integrin inhibitors and their method of use) describes multiple classes of chemical compounds which can be used as integrin inhibitors.
Another example of pharmacological agent used for cell mobilization is a blocker of CXCR4 receptor under the trademark name AMD3100 (USPTO trademark serial number 78367683). AMD3100, or Plerixafor is pharmaceutical and medicinal preparations for the treatment of HIV, inflammation, arthritis, asthma, cancer, cell transplants and cell transplant rejection, organ transplants and organ transplant rejection, angiogenesis, multiple sclerosis, bacterial infection, peripheral blood stem cell mobilization, cardiovascular disease, leukemia, drug-induced anemia, retrovirus, hematopoietic deficit resulting from chemotherapy or radiation therapy, and elevation of white blood. AMD3100 is a partial antagonist of the alpha chemokine receptor CXCR4. The CXCR4 alpha-chemokine receptor and one of its ligands, SDF-1, are important in hematopoietic stem cell homing. Plerixafor has been found to be a strong inducer of mobilization of hematopoietic stem cells from the bone marrow to the bloodstream as peripheral blood stem cells Cashen, A. F.; Nervi, B.; Dipersio, J. (2007). “AMD3100: CXCR4 antagonist and rapid stem cell-mobilizing agent”. Future Oncology 3 (1): 19-27.
Apart from pharmacological factors, physical factors are also capable to affect homing and release of stem cells from their niches. Most convenient practical examples of such physical factors are ultrasound and electromagnetic fields. Ultrasound is cyclic sound pressure with a frequency greater than the upper limit of human hearing (approximately 20 kHz). The ultrasound is used to penetrate a medium and measure the reflection signature or supply focused energy. The reflection signature can reveal the inner structure of the medium, and is usually used in medicine for diagnostical purposes (sonography). Ultrasound also has therapeutic applications (to treat stone diseases of internal organs—lithotripsy). High Intensity Focused Ultrasound is used ultrasound to ablate tumors or other tissue non-invasively in which lower frequencies than medical diagnostic ultrasound is used (25-200 kHz), but higher time-averaged intensities. Delivering chemotherapy to cancer cells and other tissues is called acoustic targeted drug delivery which uses high frequency ultrasound (1-10 MHz) and a range of intensities 0-20 W/cm2. The acoustic energy is focused on the tissue of interest to agitate its matrix and make it more permeable. Additional physiological effects of low-intensity ultrasound have been used to stimulate bone-growth and its potential to disrupt the blood-brain barrier for drug delivery. Low intensity pulsed ultrasound is used for therapeutic tooth and bone regeneration. Ultrasound in the low MHz range in the form of standing waves is a new approach to achieve a tool for separation of cells in tissues and culture, concentration and directional movement of small particles and cells. This method has specific name “acoustophoresis”. The basis of “acoustophoresis” is the acoustic radiation force, a non-linear effect which causes particles to be attracted to either the nodes or anti-nodes of the standing wave depending on the acoustic contrast factor, which is a function of the sound velocities and densities of the particle and of the medium in which the particle is immersed. U.S. Pat. application Ser. No. 20070065420 (Ultrasound therapy resulting in bone marrow rejuvenation) describes a method and system for treating a patient to repair damaged tissue which includes exposing a selected area of bone marrow of a patient to ultrasound waves or ultra shock waves so that cells comprising stem cells, progenitor cells or macrophages are generated in the area of the bone marrow of the patient due to the ultrasound, converting the cells from the bone marrow of the patient and reducing the damaged tissue in the bone marrow of the patient by repairing the damaged tissue.
An electromagnetic field is a physical field produced by the motion of electrically charged objects. It affects the activities of charged particles and objects in the vicinity of the field. Magnetic fields arise from the motion of electric charges. Low-frequency electric fields influence the human body same as they influence any other material made up of charged particles. They cause current to flow through the body to the ground. Low-frequency magnetic fields induce circulating currents within the human body. The strength of these currents depends on the intensity of the outside magnetic field. If sufficiently large, these currents could cause stimulation of cells or affect other biological processes. Heating is the main biological effect of the electromagnetic fields of radiofrequency fields. Therefore, ultrasound and electromagnetic fields represent additional approach for HSCs mobilization.
HSCs and hematopoietic progenitor cells (HPCs) are widely used for transplantation treatment of blood cell disorders. Bone marrow, peripheral blood, and umbilical cord blood (CB) currently serve as sources of HSCs for transplantation, but the demand for HLA-matched stem and progenitor cells exceeds the supply; less than 50% of patients today are able to obtain needed allogeneic transplantations. As less than 30 percent of potential recipients have HLA-identical siblings, transplantation of allogeneic HSCs is widely used. Allogeneic transplantation of bone marrow or HSCs-enriched peripheral blood often results in a severe adverse immunologic response. Umbilical cord blood is a potential source of HSCs and HPCs, but limited numbers of HSCs per CB unit limit the use of CB for transplantation to small children. Alternative sources of HSCs are, therefore, in high demand for treatment of adults.
Cells isolated from umbilical cord—anatomical structure which connects a baby with placenta—have been described in several patents. Messina et al., (U.S. Pat. No. 7,524,489 “Regeneration and repair of neuronal tissue using postpartum-derived cells”), teaches method of treatment patients with cells derived from umbilical cord which do not express CD117 while expressing oxidized LDL receptor1, interleukin 8 or reticulon 1. Mistry et al., (U.S. Pat. No. 7,510,873 “Postpartum cells isolated from umbilical cord tissue, and methods of making and using the same”) teaches method of isolation of a cell from umbilical cord by enzymatic digestion that does not express CD117, CD31, CD34, CD141 or CD45 and express CD10, CD13, CD44, CD73, CD90, PDGFr-alpha or HLA-A, and further teaches use of these cells for treatment of retinitis (Mistry et al., U.S. Pat. No. 7,413,734 “Treatment of retinitis pigmentosa with human umbilical cord cells”). Harmon et al. (U.S. Pat. No. 7,560,276 “Soft tissue repair and regeneration, using postpartum-derived cells”) teaches use of these cells from umbilical cord and their products for soft tissue repair. Davies et al., U.S. Pat. No. 7,547,546 “Progenitor cells from Wharton's jelly of human umbilical cord” teaches obtaining cells from umbilical cord and their use in tissue repair. Use of proteolytic enzymes required for all the above described methods, first, dramatically reduces cell viability and yield of colony-forming unit cells, and, second, eliminates expression of many stem cell markers on cell surface, thus not allowing using sorting techniques for isolation of stem cells. The above described methods to obtain cells from umbilical cord feature same problems as obtaining stem cells from other low volume sources—yield of viable colony-forming unit cells from this source is very low.
Hariri (U.S. Pat. No. 7,045,148) reports that the first collection of blood from the perfused placenta, referred to as cord blood, contains populations of hematopoeitic progenitor cells which are CD34 positive and CD38 positive or CD34 positive and CD38 negative or CD34 negative and CD38 positive. Subsequent perfusions of the placenta were reported to yield embryonic-like stem cells that are SSEA-3 negative, SSEA-4 negative, Oct-4 positive, ABC-p positive, CD10 positive, CD38 negative, CD29 positive, CD34 negative, CD44 positive, CD45 negative, CD54 positive, CD90 positive, SH2 positive, SH3 positive and SH4 positive. Hariri (U.S. Pat. No. 7,311,905 “Embryonic-like stem cells derived from post-partum mammalian placenta, and uses and methods of treatment using said cells”) describes a composition of human stem or progenitor cells that are positive for SH2, SH3, SH4 and Oct-4, while negative for CD34, CD45, SSEA3 and SSEA4, and obtained from placenta that has been drained from cord blood. Cells could express at least one of the following markers: CD10, CD29, CD44, CD54, CD90. Hariri (U.S. Pat. No. 7,468,276 “Placental stem cells”) describes the same placental stem cell population, adherent to plastic. Hariri (U.S. Pat. No. 7,255,879 “Post partum mammalian placenta, its use and placental stem cell populations”) teaches method to obtain the above described placental stem cell population by perfusing placenta via circulation with a perfusion solution containing an anticoagulant, growth factor or cytokine selected from a group consisting of a colony stimulating factor, interferon, erythropoietin, stem cell factor, thrombopoietin, an interleukin, granulocyte colony-stimulating factor, and any combination thereof, and collection of cells from perfusate. Methods of directed differentiation of these cells are described by Hariri (U.S. Pat. No. 7,498,171 “Modulation of stem and progenitor cell differentiation, assays and uses thereof”).
The main disadvantage of methods described by Hariri in the above cited patents is in the fact that long-term perfusion of placenta is required to obtained claimed cells. It is know to those skilled in arts that in most cases of placentas obtained by Caesarian section and in all cases of placentas collected following natural birth, placentas are ruptured. This precludes the possibility of the long-term perfusion, as perfusate is rapidly lost via ruptures of chorion; therefore, long-term perfusion becomes impracticable. In most cases, arteries of umbilical cord rapidly completely constrict, and as thrombosis develops in placental vessels perfusion of placenta by techniques claimed by Haririr's patents (U.S. Pat. Nos. 7,045,148; 7,255,879; 7,311,905; 7,468,276) becomes practically impossible. Most importantly, perfusion of placenta via natural circulation does not allow collecting populations of stem cells which are located in stroma, interstitial tissue or non-perfused regions of placental circulation. Therefore, placental perfusion and cells claimed by Hariri's patents (U.S. Pat. Nos. 7,045,148; 7,311,905; 7,468,276; 7,498,171) allow obtaining very restricted and limited cell populations present in placenta, which belong to pool located inside the circulatory space. It is, therefore, a subject matter of this invention to disclose novel techniques, methods and stem cell populations which could be obtained without placental perfusion.
Recently several patents described an improved way to obtain additional amounts of cord blood and different types of stem cells by perfusion of placenta. Hariri (U.S. Pat. No. 7,045,148) reports that the first collection of blood from the perfused placenta, referred to as cord blood, contains populations of hematopoeitic progenitor cells. U.S. patent application Ser. No. 20100248206 (Method of Isolating Stem and Progenitor Cells From Placenta) describes a method for cryopreserving fetal stem and progenitor cells in a mammalian placenta, the method comprising: perfusing a mammalian placenta with a perfusion solution comprising an anti-coagulant, a vasodilator, and a cryopreservative agent. Serikov et al (Human term placenta as a source of hematopoietic cells, Exp Biol Med 2009, 234:813-823) reported that the human placenta contains large numbers of CD34-expressing hematopoietic cells, with the potential to provide a cellular yield several-fold greater than that of a typical cord blood harvest. Cells from placental tissue generated erythroid and myeloid colonies in culture, and also produced lymphoid cells after transplantation in immunodeficient mice. AMD3100 was used to mobilize cells from placenta during 6-8 hour vascular perfusion, which allowed to inclearse yeaild of HSC from perfusate several-fold as compared to cord blood unit HCSs.
Procedures described by Hariri and others and termed as “perfusion” require putting catheters into umbilical vein and artery, and securing these catheters in place to avoid loss of perfusate. Such procedure which specifically includes placement of catheters into umbilical arteries requires very highly skilled professional to perform and in large number of cases is simply impossible. Perfusion requires complex and expensive set of equipment, continuous control of trained personnel and is impossible to perform in field conditions. Moreover, as most of cord blood units are collected in hospitals, the processing and storage happens at a few specialized centers. Therefore, shipment of material by mail is required. Such step in cord blood collection process makes perfusion of placenta to obtain HSCs practically impossible for large scale operations. Therefore, there is a need for a method, which will allow collection of HSCs from placental circulation without the need for perfusion of placenta. The present invention addresses this need and provides further related advantages.
It also the object of the present invention to provide a method of mobilizing HSCs from placenta utilizing physical means of ultrasound wave field application, and or pulsed electromagnetic field application.